The goal of this project is to understand how proteins which must be secreted or inserted into membranes are identified and directed to transport sites in the endoplasmic reticulum (ER) or, equivalently, the bacterial cytoplasmic membrane. The project combines biochemical and genetic methods to study the role of the signal recognition particle (SRP) and the SRP receptor in this process. Previous studies have shown that the SRP ribonucleoprotein recognizes nascent polypeptide chains bearing "signal sequences" and then catalyzes their translocation across the membrane upon interaction with the ER-bound SRP receptor. A major aim of this project is to elucidate the mechanism by which SRP recognizes signal sequences and releases them in a regulated manner. By studying this problem we expect to obtain insight into the structural features that enable a protein to possess broad substrate specificity and into the regulation of multi-step pathways. In recent work we have begun to analyze the function of E. coli homologs of SRP [comprised of a single protein ("Ffh") and a small RNA ("4.5S RNA")] and the SRP receptor ("FtsY"). This problem has been of particular interest in light of substantial evidence indicating that unlike mammalian cells, bacteria utilize SRP-independent mechanisms to target secreted proteins to the cytoplasmic membrane. To elucidate the function of the E. coli Ffh/4.5S RNA particle, we developed a novel genetic screen based on the concept of synthetic lethality to identify genes encoding possible targeting substrates. Eight genes and operons encoding polytopic inner membrane proteins (IMPs) were isolated by this method. We found that depletion of Ffh or expression of a dominant lethal allele of FtsY blocked the insertion of several of these IMPs into the cytoplasmic membrane, indicating that SRP is required for their biogenesis. Interestingly, we found that inhibition of the SRP pathway did not significantly affect the insertion of all IMPs, suggesting that the hydrophobicity of membrane spanning domains may not be sufficient to confer SRP-dependence. Consistent with previous studies, our results also showed that inhibition of the SRP pathway had no effect on protein export. Thus bacterial SRP appears to be have a more restricted function than its eukaryotic couterpart. We have also obtained evidence that the Ffh/4.5S RNA particle, like eukaryotic SRP, interacts with nascent polypeptides at a specific step of the ribosome cycle. Finally, our results suggest that the insertion of SRP substrates into the cytoplasmic membrane is catalyzed by the SecY protein translocation complex.